Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have investigated the suitability of 5'-p-fluorosulfonylbenzoyladenosine (FSBA) as an ATP site affinity probe for the canine kidney Na+, K+-ATPase. The purified enzyme is slowly inactivated by this compound in suitable buffers, losing about half of its activity over a two-hour period. The rate of inactivation is more rapid in 0.1 M KCl than in 0.1 M NaCl. Low concentrations of ATP protect the enzyme against inactivation, with half-maximal effects at 4 microM ATP in 0.1 M NaCl and 350 microM ATP in 0.1 M KCl. ADP also protects against FSBA inhibition, but AMP is ineffective when present at 100 microM levels. This pattern is consistent with the previously described nucleotide specificity of the Na+, K+-ATPase. Addition of protective amounts of ATP after inactivation has occurred does not restore enzyme activity, indicating that inhibition is irreversible. Measurement of the concentration-dependence of FSBA inactivation suggests an apparent Kd for binding of this compound well above 1 mM, the solubility limit of the analog. This finding is reinforced by the failure of 1 mM FSBA to compete effectively with ATP for the high-affinity ATP site of the enzyme. Nevertheless, attachment of the analog to this site is indicated by its ability to prevent [3H]-ADP binding in proportion to the number of sites it has inactivated. Studies with [3H]-FSBA show that about 1 mole of the analog attaches specifically to the alpha subunit per mole of enzyme inactivated. A similar amount of nonspecific labeling also occurs with negligible effect on enzyme activity. These findings suggest that FSBA may be useful in probing the topography of the high-affinity ATP binding site of the Na+, K+-ATPase and related enzymes.
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PMID:5'-p-fluorosulfonylbenzoyladenosine as an ATP site affinity probe for Na+, K+-ATPase. 626 29

Relaxation of smooth muscle cells induced by activation of beta-adrenoceptors was investigated in intact and skinned muscles of the guinea-pig mesenteric artery.1. In concentrations over 10(-7) M, isoprenaline reduced the resting tone of intact preparations and also the amplitude of K contractions. When Ca was applied after previous superfusion with Ca-free solution, the amount of Ca accumulated into storage sites was increased by isoprenaline in polarized and depolarized ([K](o) 128 mM) muscles. The amount of Ca stored increased even further when procaine and isoprenaline were applied simultaneously during store loading.2. Isoprenaline increased the concentration of cyclic AMP as determined by radioimmunoassay. Application of isoprenaline at a concentration of 10(-7) M increased cyclic AMP from 2.2+/-0.3 to 2.8+/-0.6 p-mole/mg wet weight and at 10(-6) M increased it to 4.5+/-0.8 p-mole/mg wet weight after 5 min incubation (n = 4).3. Application of cyclic AMP (3 x 10(-6) M) with cyclic AMP-dependent protein kinase (50 mug/ml.) had no effect on the pCa-tension relationship in the skinned muscles. However, an increased concentration of cyclic AMP (> 10(-5) M) suppressed the Ca-induced concentration only in the presence of protein kinase. This protein kinase (50 mug/ml.) alone had no effect on the Ca-induced contraction.4. In skinned fibres, the Ca store could be loaded by applying low concentrations of Ca. If cyclic AMP (3 x 10(-6) M) with protein kinase (50 mug/ml.) was applied during the loading procedure, the amount of Ca accumulated by the store increased if the loading solution contained 10(-6) M-Ca applied for 2 min or less, but if the loading solution was applied for 3 min, or if higher Ca concentrations were used, the presence of cyclic AMP with protein kinase decreased the store size, suggesting that a Ca-induced Ca-release mechanism was also being activated.5. In skinned muscles, accumulation of Ca into the store site in the presence of cyclic AMP (3 x 10(-6) M) with protein kinase (50 mug/ml.) was further accelerated by simultaneous applications of procaine (5 mM), as here the Ca-induced Ca-release mechanism was suppressed.6. These results indicate that activation of beta-adrenoceptors by isoprenaline increases the amount of cyclic AMP in the intact muscles, and leads to an increase in Ca accumulation into the store site. In the skinned muscles, the Ca-induced Ca-release mechanism is activated by cyclic AMP and the Ca receptor for contraction (leiotonin C or calmodulin) is somewhat suppressed. These effects of exogenously applied cyclic AMP require the presence of protein kinase. The relaxation following beta-adrenoceptor activation is more likely to involve Ca extrusion from the cell and accumulation of Ca in internal storage sites than suppression of the binding of calmodulin with the myosin light chain kinase.
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PMID:Mechanisms of relaxation induced by activation of beta-adrenoceptors in smooth muscle cells of the guinea-pig mesenteric artery. 628 50

The substrate specificity of diadenosine 5',5"'-P1,P4-tetraphosphate pyrophosphohydrolase from Physarum polycephalum for dinucleoside polyphosphates has been determined by high-performance liquid chromatography (HP-LC). Elution of a strong anion-exchange resin with a pH and ionic strength gradient of ammonium phosphate separates a series of monoadenosine and diadenosine polyphosphates. Most of the corresponding guanine nucleotides are also resolved on this HPLC system. One mole each of Ap4A and Gp4G is symmetrically hydrolyzed to 2 mol of ADP and GDP, respectively. Ap3A, Ap5A, Ap6A, and Ap4 are hydrolyzed, and in each case ADP is one of the products. Gp3G, Gp5G, Gp6G, and Gp4 are also substrates, and in each case GDP is one of the products. AMP, ADP, ATP, Ap2A, ADPR, GMP, GDP, GTP, NAD+, and NADP+ are not substrates. No hydrolysis of the cap dinucleotides m7Gp3Am and m7Gp3Cm was detected by HPLC. Diadenosine tetraphosphate pyrophosphohydrolase preparations were also assayed for adenylate kinase, nucleotide diphosphate kinase, NAD(P)+ pyrophosphohydrolase, phosphodiesterase, cyclic nucleotide phosphodiesterase, phosphatase, and ribonuclease activities. These enzymic activities were not detectable in diadenosine tetraphosphate pyrophosphohydrolase. The symmetrical hydrolysis of Ap4A and Gp4G is an unique catalytic property that distinguishes diadenosine tetraphosphate pyrophosphohydrolase from P. polycephalum from diadenosine tetraphosphate phosphohydrolases from other organisms.
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PMID:Diadenosine 5',5"'-P1,P4-tetraphosphate pyrophosphohydrolase from Physarum polycephalum. Substrate specificity. 629 57

NADPH-cytochrome P-450 reductase was purified to apparent homogeneity from detergent-solubilized guinea pig liver microsomes. The reductase had a mol. wt of 78,000 and contained one mole each of FAD and FMN. Electron transfer activity to cytochrome c was optimal at a pH of 8.0 and an ionic strength of 0.43. The results of kinetic experiments were consistent with a ternary-complex mechanism for the interaction of the reductase with cytochrome c and NADPH. Km values for NADPH and cytochrome c were 3.1 and 26.7 microM, respectively. Inhibition by NADP+ and 2'-AMP was competitive with respect to NADPH; Ki values were 12.1 microM for NADP+ and 46.7 microM for 2'-AMP.
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PMID:Kinetic properties of guinea pig liver microsomal NADPH-cytochrome P-450 reductase. 632 Oct 97

Experiments were carried out to investigate the time course of the release of catecholamine, dopamine-beta-hydroxylase (DBH) and adenine nucleotides from isolated chromaffin cells of guinea-pig adrenal gland. When the isolated chromaffin cells were incubated with medium containing acetylcholine (ACh) (0.1 mM), veratridine (0.1 mM) or scorpion (Leiurus quinquestriatus) venom, (10 micrograms/ml.), catecholamine was released into the medium. Catecholamine secretion induced by veratridine or scorpion venom was inhibited by tetrodotoxin (1 microM) but not by atropine (0.1 mM) plus hexamethonium (0.1 mM). On the other hand, the secretory response to ACh was abolished by the cholinergic blocking drugs but not by tetrodotoxin. DBH was released together with catecholamine into the medium in which cells were suspended with these drugs. The ratio of catecholamine (n-mole) to DBH activity (n-mole/hr) appearing in the supernatant was 7.08 +/- 0.55, 6.60 +/- 0.27 and 8.91 +/- 0.47 for ACh, veratridine and scorpion venom, respectively. These values were close to that found in the lysate of chromaffin granules obtained from guinea-pig adrenal glands (7.37 +/- 0.39). The application of ACh or veratridine to perifused chromaffin cells was found to cause a parallel increase in catecholamine and DBH secretion in the perifusion medium without corresponding amounts of phenylethanolamine-N-methyltransferase leakage. However, DBH secretion tended to last for a longer period than catecholamine secretion. Adenine nucleotides were released from perifused chromaffin cells together with catecholamine, by ACh and veratridine. ATP added to the perifusion medium was metabolized to ADP and AMP, of which the ratio (ATP, 21.6%; ADP, 34%; AMP, 17.9%) was close to those of adenine nucleotides released from the cells. The secretion of adenine nucleotides induced by both secretagogues ceased much faster than the catecholamine secretion, so that molar ratio of catecholamine to adenine nucleotides was gradually increased during and after stimulation. The results indicate that catecholamine secretion is accompanied with a simultaneous release of DBH and ATP from adrenal chromaffin cells. Therefore, it is suggested that the delayed output of DBH, unlike catecholamine secretion, in perfused adrenal glands results from the presence of a diffusion barrier for this protein. The releasable secretory granules of isolated chromaffin cells are suggested to be heterogeneous with respect to the ratio of catecholamine to ATP.
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PMID:Time course of release of catecholamine and other granular contents from perifused adrenal chromaffin cells of guinea-pig. 662 Jan 78

Soluble complexes of poly (U) and adenylic nucleotides in NaCl solutions were studied by scanning microcalorimetry. The melting enthalpies, delta Hm, of poly (U) complexes with adenosine, 2',3' -cAMP, 2'(3')-AMP, 5-AMP, ADP, ATP in 1 M NaCl are 50.5; 45.0; 42.9; 28.6; 26.1 and 25.6 kJ/mole triplets, respectively. Delta Hm is independent of the complex melting temperature, Tm. The calorimetric enthalpies are considerably lower than the apparent delta Hv.H. obtained from Tm dependence on free monomer concentration. The enthalpy of complex formation in 1 M NaCl depends neither ob the number nor on the degree of ionization of the phosphate groups but is essentially determined by their 5' - or 2'(3')-position. In contrast to 2'(3')- AMP. 2 poly (U), delta Hm of 5'AMP. 2 poly (U) increases considerably at lowering Na+ concentration. The enthalpy of poly (U) double helix melting in 1 M NaCl is 8.8 kJ/mole pairs which is 2.5 times lower than that in MgCl2 solutions.
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PMID:Calorimetric study of the complexes between polyuridylic acid and adenylic nucleotides. 730 78

The mammalian pineal organ contains photoreceptor-specific proteins, whose distribution shows conspicuous variation among different species of mammals. Nevertheless, the following general conclusions can be drawn: immunoreactions for S-antigen and recoverin labeled more pinealocytes than the rod-opsin immunoreaction. The intensity of the recoverin- and S-antigen immunoreactions varied from cell to cell. alpha-Transducin immunoreaction was absent from the pineal organ of all mammals investigated with the exception of the blind mole rat. Immunoreaction for the cyclic GMP-gated cation channel was undetectable in the pineal organ of all mammals investigated. The functional significance of photoreceptor-specific proteins in the mammalian pineal organ remains unknown. It has been speculated that the S-antigen might be involved in adrenergic transduction mechanisms. To test this assumption, we have started to analyze calcium responses of single rat pinealocytes to norepinephrine stimulation using the Fura-2 technique. The cells were subsequently labeled by means of S-antigen immunocytochemistry. These combined investigations showed that variation in S-antigen immunoreactivity is not correlated with differences in the rapid calcium response to stimulation with norepinephrine. It remains to be determined whether cells displaying different intensities of the S-antigen immunoreaction show different cyclic AMP responses to noradrenergic stimulation. Investigations along this line should help to clarify further whether there is indeed a relation between the expression of S-antigen and noradrenergic transduction mechanisms in the mammalian pineal organ.
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PMID:Photoreceptor-specific proteins in the mammalian pineal organ: immunocytochemical data and functional considerations. 752 60

Salicylate hydroxylase from Pseudomonas putida S-1 was irreversibly inactivated by trinitrobenzenesulfonic acid (TNBS). The reaction was linearly dependent on TNBS concentration and the second-order rate constant was 120 M-1.min-1 for the holoprotein at pH 8.5. Modification of one mole of lysine residue per mole of enzyme caused a large loss of the activity, and the enzyme was no longer able to show NADH-dehydrogenase activity after uncoupling. The presence of NADH, NAD+, ATP, or AMP afforded protection against the inactivation. The enzyme modified at a single lysine residue was isolated by hydrophobic chromatography as an apoprotein form and characterized. It could bind FAD with the same Kd value for that of native apoprotein. The apparent Michaelis constant of the enzyme was increased 13-fold for NADH, but not for salicylate. Vmax for NADH oxidation was decreased to one-fifth of that of the native enzyme. A peptide containing one trinitrophenyl-lysine residue was isolated from the chymotryptic digest of the modified enzyme and its amino acid sequence was determined to be TADVAIAADGIKSSM, which is homologous to the sequence from R-154 to I-168 of salicylate hydroxylase from P. putida PpG7. The lysine in the peptide may represent a basic residue interacting with an anionic group of NADH in the binding site of the enzyme.
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PMID:Identification of a lysine residue in the NADH-binding site of salicylate hydroxylase from Pseudomonas putida S-1. 762 25

Fluorescent 2'-O-dansylated (DANS) purine nucleotides were synthesized. The fluorescence of the nucleotide derivatives is quenched in aqueous solutions but strongly enhanced on binding to the uncoupling protein (UCP) from brown adipose tissue mitochondria. The fluorescence enhancement was 30-, 10-, and 10-fold for DANSGTP, DANSATP, and DANSADP. One mole of DANS nucleotide binds to 1 mol of dimeric UCP. The binding affinity ranges from 10(5) to 10(8) M-1, similar to that of the unsubstituted nucleotides, while dansylation of AMP increases the affinity 50-fold. The pH dependence in the pKD/pH plots for the DANS nucleotides is basically similar to that for the unsubstituted nucleotides, i.e., for nucleoside diphosphates the slope delta pKD/delta pH < -1 at pH 5-6.5, = -1 at pH > 6.8, and only for triphosphates = -2 at pH > 7.2. Two different protonation sites with a pKH approximately 4 (Asp/Glu) and pKH approximately 7.2 (His), only for nucleoside triphosphates, are suggested to be involved in binding. The higher affinity of DANSGTP indicates additional participation in binding of the C-6 oxygen on the guanine. The binding as measured with the anion exchange method agrees with the fluorescence measurement for DANSGTP, whereas for the more loosely binding DANSATP it is 40% lower. This is interpreted in terms of tight/loose UCP-nucleotide complexes, 100% tight complex for DANSGTP (as well GTP or ATP) but 40% loose complex for DANSATP. By measuring the rapid kinetics using the fluorescence signal, the binding rate is found to be fast and fairly constant for the various nucleotides, whereas the dissociation is slow and strongly nucleotide dependent. The rates are pH dependent with delta pkon/delta pH = 1 for all the nucleotides and delta pkoff/delta pH = -1 for DANSNTP but more weakly with delta pkoff/delta pH < -0.5 for DANSADP and DAN-ATP. The pH dependence of the binding rate corresponds to a protonation at the carboxylate group (Glu/Asp). The high pH dependence of the dissociation rate only for DANSNTP is explained by deprotonation at the HisH+ which is involved only in nucleoside triphosphate binding. This is in line with the very strong pH dependence of nucleoside triphosphate affinity above pH 7 with a delta pKD/delta pH = -2 as an important regulatory mechanism for the H+ transport activity of UCP. The differences of the DANS nucleotides versus the DAN and unsubstituted nucleotides as well as the nucleoside tri- versus diphosphate are rationalized in a specific H+ dependent regulatory mechanism at the binding site.
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PMID:Fluorescent nucleotide derivatives as specific probes for the uncoupling protein: thermodynamics and kinetics of binding and the control by pH. 781 18

The vitamin D hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is generated by a series of hydroxylation steps in the liver and kidneys. We investigated whether naturally vitamin D-deficient subterranean mammals (naked mole rats, Heterocephalus glaber) employ the same enzymatic pathways, and whether these are regulated in a similar manner to that established for other mammals. Vitamin D3-25-hydroxylase in the liver and both 25-hydroxyvitamin D3-1-hydroxylase and 25-hydroxyvitamin D3-24 hydroxylase (1-OHase and 24-OHase) in the kidney were detectable in mole rats. As expected for vitamin D-deficient mammals, the 1-OHase activity predominated over the 24-OHase. After mole rats received a supraphysiological supplement of vitamin D3, 1-OHase activity was suppressed and 24-OHase activity was enhanced. Irrespective of vitamin D status, forskolin (a protein kinase A activator) and dibutyryl cyclic AMP did not alter the activity of either 1-OHase or 24-OHase. These findings suggest that the response of renal hydroxylases to parathyroid hormone was blunted. Phorbol esters, 12-O-tetradecanoylphorbol 13-acetate (TPA) and 1-oleoyl-2-acetylglycerol (OAG) (protein kinase C activators), suppressed 1-OHase activity. 24-OHase activity was induced by TPA but not by OAG. These effects were similar to those illicited by vitamin D3 supplementation but were additive in that they increased the responses shown in vitamin D-replete mole rats. These data confirm that naturally vitamin D-deficient mole rats can convert vitamin D3 to the hormone, 1,25(OH)2D3.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Vitamin D hydroxylases and their regulation in a naturally vitamin D-deficient subterranean mammal, the naked mole rat (Heterocephalus glaber). 785 93


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